PROJECT 3 ABSTRACT: Major depressive disorder (MDD) remains one of the most prevalent and costly medical disorders. A third or more of patients may not achieve symptomatic remission despite multiple medication treatments; many other individuals are simply unable or unwilling to initiate prescription pharmacologic or psychosocial treatment. Complementary and alternative medications (CAM) represent an important option for such patients. In addition to understanding which CAM antidepressant strategies are efficacious for depression as well as for stress, the aim of Project 1, it would be highly valuable to understand the mechanisms by which certain CAM treatments exert their therapeutic effect. This understanding could increase the acceptability of current treatments, allow better matching of patients with effective treatments, and facilitate the investigation and development of novel CAM treatments. For standard antidepressant treatments, multiple hypotheses regarding mechanisms of action have been developed. These include (i) promotion of neuroplasticity, (ii) modulation of inflammation, and (iii) promotion of neurogenesis. To date, investigation of these latter hypotheses has been hampered by a lack of direct models of human neurobiology ? and particularly neuropathology - amenable to rapid screening and quantitative functional assessment. That is, it has not been possible to examine whether these hypotheses are supported in neural tissue from patients with the particular disease targeted by these interventions. Progress in stem cell technology and developmental neurobiology allows a novel strategy that forms the focus of Project 3. Dermal fibroblasts from 180 patient participants in the randomized trial of Project One will be reprogrammed (transdifferentiated) to induced neurons (iNs). Transcriptional profiles of these iN's will be compared after exposure to n-3 fatty acids, S- adenosyl L-methionine (SAMe), or vehicle to test whether the two CAMs regulate genes related to neuroplasticity (Aim 1a), and whether degree of modulation of neuroplasticity is associated with treatment efficacy. In parallel, a subset (10 per treatment arm) of patient-derived fibroblasts will be reprogrammed to induced pluripotent stem cells. These cell lines will then be differentiated into neuronal precursors and ultimately to mature neurons. Work by this group and others indicates that it is possible to generate such cells and incorporate them in high-throughput, quantitative functional assays to characterize phenotypes relevant to antidepressant mechanism. Specifically, the hypothesis that n-3 fatty acids and SAMe modulate inflammatory markers on neural-lineage cells (Aim 2a), and promote neurogenesis (Aim 2b), will be tested using validated assays. The hypothesis that these mechanisms are associated with treatment efficacy will also be tested. In addition to examining these primary hypotheses, this project will establish a critical resource for future investigation of CAM compounds, a biobank of 180 fibroblasts and 30 pluripotent stem cells and neuronal precursor cells, all derived from patients with MDD participating in Project 1's placebo-controlled investigations. NARRATIVE Major depressive disorder is a major contributor to morbidity worldwide, and existing treatments fail to yield symptomatic remission in ~1/3 of patients. While Complementary and Alternative Medicine (CAM) compounds are increasingly used to treat depression, and well-accepted by patients, their mechanisms of effect have not been fully characterized. The proposed investigation will test specific hypotheses extending preliminary data using unique patient-derived stem cells and induced neurons, while at the same timing establishing biomarkers, biosignatures, and a biobank to facilitate future CAM studies.